Long term goals are to understand the physiological regulation of cardiac muscle contraction, particularly Ca and E-C coupling. We will use a number of techniques and protocols to study in detail the regulation of [Ca]i via individual Ca transport mechanisms (e.g. voltage clamp (perforated and ruptured patch), indo-1 fluorescence, digitonin permeabilization, Ca minielectrodes and some subcellular fractionation). The emphasis is on quantitative understanding of how certain Ca transporters are regulated in the intact isolated ventricular myocyte (e.g. SR Ca release channel, SR Ca-pump and Na/Ca exchange) and how these systems interact with each other to regulate SR Ca content and the fraction of Ca released. Specific aims for the next period will be to: 1) Develop equilibrium/null point methods for in situ calibration of cytoplasmic [Ca]. 2) Measure the free intra-SR [Ca] in ventricular myocytes. 3) Measure "normal" and maximal SR Ca content in intact ventricular myocytes. 4) Evaluate the regulation of the fraction of SR Ca released during E-C coupling. 5) Evaluate the rate of resting leak of Ca from the SR. 6) Determine how calmodulin and CaMKII alter Ca regulation in ventricular myocytes. 7) Determine the molecular basis for the unique Ca extrusion properties in ferret ventricle. 8) Determine the quantitative role of Ca influx via Na/Ca exchange in normal E-C coupling. These studies will provide important new fundamental quantitative information concerning cellular Ca fluxes and cardiac E-C coupling. In addition, certain agents, such as Calmodulin and Ca-Calmodulin dependent protein kinase (CaMKII) are known to modify multiple Ca transport processes when studied in isolated systems. Our studies will emphasize how these (and some other) agents work in the cellular environment and will provide quantitative insight into how the multiple systems interact dynamically in the regulation of cell Ca. Cellular Ca fluxes are in a dynamic, yet delicate balance in heart and this will be studied in detail. The consequences of disturbing this balance can include contractile failure, spontaneous contractions (i.e. arrhythmogenic) and impaired relaxation. Any of these dysfunctions will compromise the ability of the heart to function effectively as a pump. Results from the focused aims above will contribute to our comprehensive overall understanding of Ca regulation in the heart.